Higher animals have a system for supplementing cells after death of cells that form tissues and organs by apoptosis or injury. For example, the amphibian newt can regenerate the limbs and tail if they are cut off, and birds can easily regenerate the nervous system. Mammals have lost such high regenerative capacity, but their liver can regenerate oneself unless it has suffered a severe damage. Additionally, skin, hair, small intestine and hematopoietic cells are regenerated while a mammalian individual is alive. In these tissues, the cell cycle of new cell birth, differentiation and death is repeated as long as the individual is alive. The regenerative capacity depends on cells known as stem cells (Fuchs and Segre, Cell, 100, 143-155, 2000; Weissman Cell, 100, 157-168, 2000). Of various cells types forming tissues and organs, blood cells, nerve cells, vascular endothelial cells and epithelial cells are known to mature through several stages from the undifferentiated cells known as stem cells.
Stem cells have the ability of self-replication (self-renewal) to reproduce oneself by cell division, and the ability of differentiation into specific mature cells. In stem cells, a delicate balance is struck between the self-replication and the differentiation.
For one mechanism of maintaining the balance, the hereinafter-mentioned systems have been proposed.
The localization sites of stem cells are referred to as niches, in which there is a molecular infrastructure that allows the stem cells to be maintained and reproduced. In niches, the stem cells are typically maintained in a growth arrest phase. Once released from the arrested conditions due for example to a tissue injury, the stem cells enter a growth phase, and form a certain population of cells. In this growth process, heterogeneity arises within the population of cells. Some cells re-enter the cell arrest phase and retain their characteristics as stem cells, whereas others statistically express a transcription factor and thereby become destined to differentiate, and subsequently differentiate to different lineages of mature cells. There are thought to be stromal cells in the niches, which can come in contact with the stem cells and trigger a signal for growth arrest in the stem cells.
In the differentiation process to mature cells from so-called precursor cells (or progenitor cells), wherein the precursor cells has left the niches, expressed the transcription factor, and thereby become destined to differentiate, there is a mechanism which controls the process to allow the differentiation to proceed properly. It is thought that other stromal cells, which are different from the stromal cells that trigger a signal for growth arrest in the stem cells, come in contact with the precursor cells, and the precursor cells are subjected to control by certain molecules expressed by these stromal cells, and then that differentiation proceeds properly.
If the identities of the stem cell growth activating/arresting signals possessed by the stromal cells (or the stromal cells population) present in the niches, or those of the precursor cell differentiation control signals, are ascertained, whereby the methods for keeping stem cells in an undifferentiated state for a long time and for controlling differentiation of stem cells, by controlling the growth and arrest of stem cells, can be provided. These methods have many applications in fields such as regenerative medicine, gene therapy and transplantation. Specifically, the methods can be used in hematopoietic stem cell transplantation for a medical treatment of aplastic anemia, or in neural stem cell transplantation for a medical treatment of Alzheimer's disease, but these are only a few examples.
Various studies have been conducted in the past aimed at ascertaining the identities of stem cell growth activating/arresting signals and of precursor cell differentiation control signals.
Leukemia inhibitory factor (LIF) and transforming growth factor (TGF-β) are known to be cytokines which inhibit the differentiation of stem cells. LIF is known to cause the growth of mouse embryonic stem cells without differentiation, but it does not have such effect on mouse hematopoietic stem cells. Also, LIF does not affect human or monkey embryonic stem cells. For TGF-β, there are many reports regarding its inhibitory effects on various types of cells, but no fixed consensus has been obtained regarding its effect on stem cells. Examples of the molecules that control the differentiation of precursor cells are M-CSF, GM-CSF, G-CSF, SCF, TPO and FLK ligands. However such molecules discovered to date cannot account for the differentiation of various types of cells, which suggests the presence of hitherto unidentified molecules.
Recently, Notch, which is a molecule involved in differentiation control of nerve cells in Drosophila, has been discovered, and homologs of this molecule have been found in a broad spectrum of organisms across the classification of invertebrates and vertebrates (Artavanis-Tsakonas et al., Science 268, 225-232, 1995). In mammals, it has been shown that the mutation of Notch is related to T cell leukemia and lymphoma (Pear et al., J. Exp. Med. 183, 2283-2291, 1996). It has also demonstrated that expression of activated Notch molecule in myeloblast cell lines causes the inhibition of their innate ability to differentiate into neutrophils by G-CSF (Milner et al., Proc. Natl. Acad. Sci. USA 93, 13014-13019), and the Notch molecule are involved in the determination of the fate of CD4/CD8 cells in T cell differentiation (Robey et al., Cell 87, 483-492, 1996). Therefore, Notch molecules have attracted further attention as differentiation control molecules. Moreover, Delta and Serrate, which are ligands of the Notch molecule, have been identified in Drosophila (Kopczynski et al., Genes Dev., 1723-1753, 1988, Thomas et al., Development, 111, 749-761, 1991). X-Delta and D111, homologs of Delta, have been identified in the Xenopus (Chitnis et al., Nature, 375, 761-766, 1995) and mouse (Bettenhausen et al., Development 121, 2431-2418, 1995), respectively, and Jagged, homologs of Serrate, has been identified in rat and human (Luo et al., Mol. Cell. Biol. 17, 6057-6067, 1997).
From these findings, Notch receptor, and ligands thereof (Delta, Serrate and Jagged), are now attracting attention as cell differentiation and growth control molecules.
Comparing the structures of Notch, Delta and Jagged, the repetition of an EGF (Epidermal Growth Factor)-like domain is commonly found in them (Lindsell et al., Cell, 80, 909-917, 1995). The repetition is referred to as EGF-like repeat sequence or EGF-like repeat motif.
The consensus sequence of the EGF-like domain is C-X-C-X(5)-G-X(2)-C (SEQ ID NO:32) or C-X-C-X(2)-[GP]-[FYW]-X(4, 8)-C (SEQ ID NOS: 33 and 34). These domain structures are found in EGF and many extracellular proteins, and are involved in protein interactions or cellular interactions (Campbell and Bork Curr. Opin. Struct. Biol, 3, 385-392, 1993, Rao et al., Cell, 82, 131-141, 1995).
These suggest that the stromal cells in the niches possess a differentiation and growth control molecule, and the molecule belongs to the Notch, Delta and Jagged family. But the previously identified molecules of the family cannot explain the differentiation and growth control mechanism of stem cells. Accordingly, it is thought that there is also a hitherto unidentified functionally similar molecule as the above molecule possessed by the stromal cells.
A transcriptional induction system is known as a common gene expression control mechanism in animals (Nature, 321: 409-413, 1984). A promoter is generally located 5′ upstream to a region that is transcribed into mRNA on a chromosome. Furthermore, through binding or dissociation of a transcription factor to a sequence referred to as the regulatory region within the promoter sequence (transcriptional regulatory sequence), the promoter regulates the transcription level of a gene that is present in the 3′ downstream region of the promoter. Therefore, the gene expression level at the transcription stage can be estimated to some extent by measuring promoter activity. In the meantime, promoter activity is not affected in most cases by the 3′ downstream region thereof. Hence, promoter activity can be measured by inserting an appropriate reporter gene encoding an enzyme protein or the like into a downstream region of the promoter and then detecting the expression of the reporter gene. Very sensitive and convenient promoter activity measurement has become possible with the use of such a reporter, owing to recent technical innovation. Thus, such promoter activity measurement is used for drug screening and examination of biological functions. For example, screening with the promoter of peroxisome proliferator activated receptor γ (PPARγ) that is a transcription factor for adipose cells differentiation, for a compound that controls the expression of PPARγ was reported (Cell, 99: 239-242, 1999).
Production of transgenic non-human animals using promoters has also been performed. In general, it is difficult to examine the functions of genes that are essential for developmental processes or maintenance of living systems, because deletion of such genes is often lethal in mice. Conditional gene targeting techniques have been used as a potential method for addressing the problem, using a Cre-loxP recombination system under control of a promoter.
Cre recombinase is a site-specific recombinase derived from bacteriophage P1 and specifically recognizes a loxP sequence of 34 base pairs. This enzyme mediates recombination between two loxP sequences, and then a DNA fragment flanked by the two loxP sequences is excised in a cyclic form only under conditions where Cre recombinase is expressed, and the DNA fragment is deleted. For example, lck is a gene that is expressed in T cells and is strongly expressed particularly in the thymus where the development and differentiation of T cells take place. Thus, in a mouse in which a Cre recombinase gene ligated to downstream of the promoter of the lck gene has been introduced, Cre recombinase is specifically expressed only in T cells and the gene flanked by loxP sequences is disrupted (Science, 265: 103-106, 1994, Proc. Natl. Acad. Sci. U.S.A., 92: 12070-12074, 1995).
Mice known to have a Cre recombinase gene under control of such a tissue-specific promoter used therein includes: a mouse having a PO promoter that is expressed in neural crest cells (Dev Biol, 212: 191-203, 1999); a mouse having an L7 promoter that is expressed in Purkinje cells (Genesis, 28, 93-8, 2000); a mouse having a keratin 14 promoter that functions in epidermal basal cells (Horm Res, 54: 296-300, 2000); a mouse having an Mx1 promoter whose activity is induced in the presence of interferon (Science, 269: 1427-1429, 1995); and a mouse having a crystallin promoter that functions in the lens of the eyes (Proc. Natl. Acad. Sci. U.S.A., 89: 6232-6236, 1992). Discovery of a new tissue-specific promoter in addition to these promoters may cause further advancement in functional verification of genes by the conditional gene targeting.
Promoters are important also in production of recombinant proteins. When a protein is recombinantly produced using cells, the gene of a target protein is ligated downstream of a promoter and then the resultant is introduced into and expressed by cells. When animal cells are used as hosts, in general, promoters derived from viruses, such as SV40 and CMV (Proc. Natl. Acad. Sci. U.S.A., 78: 1527-1531; 1981, Nature, 329: 840-842, 1987), an actin gene promoter (Gene, 108: 193-200, 1991), and an elongation factor gene promoter (Nucleic Acids Res., 18: 5322, 1990) are used. However, the strength of the activity of these promoters differs depending on the types of proteins to be expressed and host cell types. Hence, it is necessary to examine such combination to select an optimum promoter. Therefore, provision of a new promoter is always desired for more effective production of individual proteins.